The entire route in Florida runs within Escambia County. From its terminus north to State Road 296, it is known as North Palafox Street. From this point it is known as Pensacola Boulevard north to Ten Mile Road, approximately one mile north of U.S. Route 90 Alternate. Between SR 296 and the Molino community, U.S. 29 runs parallel to its former routing, which is now County Road 95A. This former routing continues the name North Palafox Street from SR 296 north to Ten Mile Road.

U.S. Highway 29 and SR-15 traverse Alabama in a general northeast/southwest slope. It has never been a major route in the state; its significance was completely overshadowed with the completion of Interstate 65 and Interstate 85 during the 1970s. Today, US-29 and SR-15 serve primarily to connect numerous smaller towns and cities in the southwest, south-central, and eastern parts of Alabama, notably passing through Auburn University, and near Troy University, and Tuskegee University in the east.

US Highway 29 no longer passes through downtown Auburn or downtown Opelika. The US Highway is concurrent with Interstate Highway 85 from Exit 51, south of Auburn, to Exit 62, north of Opelika. This change was made by Alabama Department if Transportation in the 1990s. Route markers have been appropriately relocated since then.

US 29 was built as the main highway between Greenville and the other city of northwestern South Carolina, Spartanburg. The construction of Interstate 85 connecting Greenville to Spartanburg left US 29 underused until recent decades.

US 29 enters Washington, D.C., via the Francis Scott Key Bridge adjacent to Georgetown University. The designation turns east onto the Whitehurst Freeway, bypassing Georgetown to the south. Upon crossing Rock Creek, the freeway ends, becoming the at-grade K Street. US 29 remains on K Street to 11th Street, where US 29 turns north onto 11th for seven blocks. At Rhode Island Avenue, US 29 turns right. US 29 northbound turns left at 6th Street NW (touching US 1 where it turns from Rhode Island Avenue to 6th Street); it follows 6th Street NW for two blocks and then turns left onto Florida Avenue NW, where it then turns right onto Georgia Avenue NW. US 29 southbound at this point, however, follows 7th Street, NW to Rhode Island Avenue NW. The route maintains a northerly routing as it passes through northern Washington, D.C. and enters Maryland. During its alignment with Georgia Avenue NW, US 29 bypasses the Howard University campus to the west.

Warrenton Turnpike is the former name of US 29 through Prince William County, Virginia. This is the name that was used for this road during the Civil War. Although the road has been expanded past Manassas into four lanes, it remains a rural two lane highway through Manassas National Battlefield Park, where Interstate 66 carries through traffic. On either side of the road through the battlefield, split rail fences define property borders. The route has also been called the Bill-Beth Highway as it was the name of the children of the original highway developer.